Epidermal Growth Factor (EGF) and it anlogs represent a family of polypeptides having a variety of biological activities. Human EGF itself is a 53 amino acid polypeptide. Its analogs vary in the number of aminot acids in the polypeptide chain. A variety of these have been described in the literature. For example, see U.S. Pat. No. 3,917,824 issued Nov. 4, 1975. The literature has also described various biological activities for these materials. Each material may or may not have the same activity or as broad a biological activity as the others, but in general, it has been found that EGF and its analogs inhibit the secretion of gasric acid and promote cell growth. Thus they have been useful in wound healing applications.
Human EGF is found in the urine of young males, in the maxillary glands, and in various other locations throughout the body. Present techniques for producing human EGF and its analogs largely involve isolation of the active components from urine, and to a lesser extent, production using recombinant DNA methods.
The difficulty inherent in the first of these is quite apparent. Isolation from urine sources is time consuming, expensive, and dependent on the supply of raw material. Furthermore, the isolation of intact human EGF is complicated by the presence of closely related analogs. Current procedures leading to a recombinant method for producing EGF have not been entirely satisfactory because of apparent instability of human EGF during production and purification. Some of the disadvantages will become more apparent as more detail is described in this specification.
EGF, also known as urogastrone, contains 53 amino acids as shown in the following sequence:
______________________________________ AsnSer Asp Ser Glu Cys Pro Leu Ser HisAsp GlyTyr Cys Leu His Asp Gly Val Cys MetTyr IleGlu Ala Leu Asp Lys Tyr Ala Cys AsnCys ValVal Gly Tyr Ile Gly Glu Arg Cys GlnTyr ArgAsp Leu Lys Trp Trp Glu Leu Arg ______________________________________
The above formula is the formula for EGF as it exists in humans and as reported in the literature. The invention as described here relates to the microbial production of human EGF and some of its biologically active analogs. However, it is equally applicable to mouse EGF and in fact any EGF which has an equal or smaller number of glutamyl residues than human EGF.
It is to be noted in the sequence shown above that residues 5, 24, 40 and 51 are glutamyl. The molecule in its natural form is folded such that there are disulfide linkages between residues 6-20, 14-31, and 33-42.
While it is highly desirable to produce this material in recombinant DNA systems employing E. Coli, there has been a significant obstacle to overcome because the E. coli tends to produce the EGF in its reduced form which is not stable in the presence of endogenous bacterial proteases. It has been reported in the literature that to increase the stability, one should employ a leader sequence to produce an insoluble fusion protein protein readily recoverable from the cell paste. The selection of the specific leader sequence is known to be difficult. At the end of the polypeptide isolation phase, the leader sequence must be separated and digested away from the EGF moiety at the N terminal amino group thereof. Even when an appropriate leader sequence is employed, great difficulty has been encountered in purifying the resulting polypeptide. It is often the case that tedious chromatographic separations are required leading to a loss of product.